Ultrasensitive Electrochemistry by Radical Annihilation Amplification in a Solid-Liquid Microgap.

We report a technique to amplify the electrochemical signal within micro- and nanodroplets via radical annihilation amplification. Toluene droplets filled with decamethylferrocene (DmFc) are suspended in an aqueous solution containing 10 mM NaClO4 and 10 µM Na2C2O4. When a toluene droplet irreversibly collides with an ultramicroelectrode biased sufficiently positive for concurrent oxidation of DmFc and oxalate (C2O42-), blip-type responses are observed in the amperometric i-t trace even when the concentration of DmFc is 50 nM. The toluene droplet wetting the ultramicroelectrode effectively creates a microgap, where DmFc molecules are oxidized to DmFc+. In the continuous phase, the oxidation of oxalate (C2O42-) produces a strong reducing agent, CO2•-. Regeneration of DmFc via radical annihilation amplifies the current, similar to conventional nanogap experiments. This experiment allows one to observe the electrochemistry of hundreds to thousands of molecules trapped in a femtoliter droplet, enhancing the sensitivity of droplet-based electrochemistry by 5 orders of magnitude. Finite element simulations validate our experimental results and indicate the importance of the droplet geometry to amplification.

Quantifying Interferent Effects on Molecularly Imprinted Polymer Sensors for Per- and Polyfluoroalkyl Substances (PFAS).

Per- and polyfluoroalkyl substances (PFAS) are emerging as harmful environmental micropollutants. Generally, PFAS species are quantified by mass spectrometry, for which a collected sample is taken to a centralized facility. Robust techniques to quantify PFAS in the field are necessary to diagnose environmental contamination at the earliest onset of pollution. Here, we developed a molecularly imprinted polymer (MIP) electrode for the detection of perfluorooctanesulfonate (PFOS) and explored the MIP surface and the effects of interfering molecules. MIPs were formed by the anodic deposition of o-phenylenediamine (o-PD) in the presence of PFOS template molecules on a glassy carbon macroelectrode. The performance of the resulting MIP electrode was evaluated by the current obtained from the oxidation of ferrocene carboxylic acid as the electrochemical probe. The MIP electrode was able to detect PFOS with a detection limit of 0.05 nM, which is lower than the health advisory limit of 0.14 nM reported by the U.S. EPA. To better understand PFOS association into the MIP, a Langmuir binding model was developed based on the changes in electrochemical responses of the MIP. Fitting the model to the experimental data gave an association constant (KA) of 4.95 × 1012 over a PFOS concentration range of 0 to 0.05 nM. The binding isotherm of other commonly found substances in contaminated water sources such as chloride, humic acid, perfluorooctanoic acid (PFOA), and perfluorobutanesulfonate (PFBS) was also investigated. In the case of chloride and humic acid, the calculated KA values of 9.05 × 107 and 6.01 × 105, respectively, indicate relatively weak adsorption of these species on the MIP. However, PFOA, which is the carboxylate analog of PFOS, revealed a very close KA value (3.41 × 1012) to PFOS. A greater KA value (1.43 × 1013) was obtained for PFBS, which possesses the same functional group and a smaller molecular size compared to PFOS. The presented platform emphasizes the necessity to develop new strategies to make MIP sensors more specific if practical applications are to be pursued.

Modification of IgE binding to αS1-casein by proteolytic activity of Enterococcus faecium isolated from Iranian camel milk samples.

Milk is a perfect source of nutrients for neonates. When breast feeding cannot be done, an infant's alimentation is usually initiated to cow's milk, among the primary foods. It has been reported that about 2.5% of juveniles under the age of 3 years manifest allergic reactions to cow's milk proteins. Among the cow's milk proteins, casein fractions are considered as the strongest allergenic proteins. The proteolytic enzymes of lactic acid bacteria (LAB), during fermentation of dairy products, can break down milk proteins especially caseins and subsequently reduce the immune reactivity of allergenic proteins. In this research, raw bovine and camel milk samples were screened for cocci LAB strains and after isolation, their proteolytic activity against bovine milk caseins were evaluated by SDS-PAGE and RP-HPLC. The potential of cocci LAB strains on αS1-casein degradation and their potential to break down the principle allergenic epitopes of this protein was detected using indirect competitive ELISA. Molecular identification of the best proteolytic strain was fulfilled by 16S rDNA fragment sequencing with universal primers. The obtained results demonstrated that Enterococcus faecium isolated from raw camel milk samples was the most efficient isolate in hydrolyzing Na-caseinate and αS1-casein. Hydrolysated αS1-casein by Enterococcus faecium was also less recognized by IgE of bovine milk allergic patients' sera in comparison with native αS1-casein. It has been proposed that Enterococcus faecium could be an efficient strain in allergenicity reduction of cow's milk proteins. So it could be an excellent candidate to be potentially used in dairy industries.

Allergenicity reduction of bovine milk ß-lactoglobulin by proteolytic activity of lactococcus lactis BMC12C and BMC19H isolated from Iranian dairy products.

Nowadays health benefits of bioactive food constituents, known as probiotic microorganisms, are a growing awareness. Cow's milk is a nutritious food containing probiotic bacteria. However, milk allergenicity is one of the most common food allergies. The milk protein, ß-lactoglobulin (BLG), is in about 80% of all main cases of milk allergies for children and infants. With the aim of screening proteolytic strains of lactic acid bacteria to evaluate their potential for the reduction of allergenicity of the major bovine milk proteins, we isolated new proteolytic strains of cocci lactic acid bacteria from traditional Iranian dairy products. The proteases produced by these strains had strong proteolytic activity against BLG. Proteolysis of BLG, observed after sodium dodecyl sulfate-PAGE, was confirmed by the analysis of the peptide profiles by reversed-phase HPLC. The two isolates were submitted to 16S rDNA sequencing and identified as Lactcoccus lactis subsp. cremoris and Lactcoccus lactis subsp. hordniea. The competitive ELISA experiments confirmed that these isolates, with high proteolytic activity, reduce significantly the allergenicity of BLG. Accordingly, these isolates can reduce the immunoreactivity of bovine milk proteins, which can be helpful for the production of low-allergic dairy products.

Simultaneous Electrochemical Speciation of Oxidized and Reduced Glutathione. Redox Profiling of Oxidative Stress in Biological Fluids with a Modified Carbon Electrode.

The simultaneous electrochemical quantification of oxidized (GSSG) and reduced glutathione (GSH), biomarkers of oxidative stress, is demonstrated in biological fluids. The detection was accomplished by the development of a modified carbon electrode and was applied to the analysis of biological fluids of model organisms under oxidative stress caused by lead intoxication. Nanocomposite molecular material based on cobalt phthalocyanine (CoPc) and multiwalled carbon nanotubes functionalized with carboxyl groups (MWCNTf) was developed to modify glassy carbon electrodes (GCE) for the detection of reduced and oxidized glutathione. The morphology of the nanocomposite film was characterized by scanning electron microscopy (SEM) and profilometry. The electrochemical behavior of the modified electrode was assessed by cyclic voltammetry (CV) to determine the surface coverage (Γ) by CoPc. The electrocatalytic behavior of the modified electrode toward reduced (GSH) and oxidized (GSSG) forms of glutathione was assessed by CV studies at physiological pH. The obtained results show that the combined use of CoPc and MWCNTf results in an electrocatalytic activity for GSH oxidation and GSSG reduction, enabling the simultaneous detection of both species. Differential pulse voltammetry reveals detection limits of 100 µM for GSH and 8.3 µM for GSSG, respectively. The potential interference from ascorbic acid, cysteine, glutamic acid, and glucose was also studied, and the obtained results show limited effects from these species. Finally, the hybrid electrode was used for the determination of GSH and GSSG in rat urine and plasma samples, intoxicated or not by lead. Both glutathione forms were detected in these complex biological matrixes without any pretreatment. Our results portray the role of GSH and GSSG as markers of oxidative stress in live organisms under lead intoxication.

Stochastic electrochemistry and photoelectrochemistry of colloidal dye-sensitized anatase nanoparticles at a Pt ultramicroelectrode.

We report the stochastic interactions between dye sensitized anatase nanoparticles, suspended in a colloid, and a Pt ultramicroelectrode (UME) that result in step-wise behavior in the current vs. time response. The stochastic currents are observed in the dark and under illumination. In the dark, the currents are anodic, consistent with the oxidation of the dye N719 at the Pt surface. The electrochemical behavior of the dye was investigated in MeOH and MeCN with a quasireversible cyclic voltammogram (CV) observed at 1 V s-1. The anodic currents observed in the dark due to nanoparticles (NPs) at the Pt surface are consistent with the CVs in MeOH and MeCN. Under illumination cathodic steps are observed and assigned to the reduction of the oxidized form of the dye generated after electrons are injected into the TiO2 NPs. The colloidal behavior is a strong function of the history of the colloid with illumination time increasing the size of the agglomerates and with larger agglomerates being less photoelectrochemically active. Agglomerates of ca. 100 nm in diameter are proposed to be photoactive entities with a higher probability of detection that contribute to the staircase photocurrent response.